Exploration and optimization of substituted triazolothiadiazines and triazolopyridazines as PDE4 inhibitors

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Bioorganic & Medicinal Chemistry Letters xxx (2009) xxx–xxx

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Bioorganic & Medicinal Chemistry Letters

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FExploration and optimization of substituted triazolothiadiazinesand triazolopyridazines as PDE4 inhibitors

Amanda P. Skoumbourdis a, Christopher A. LeClair a, Eduard Stefan b, Adrian G. Turjanski c,William Maguire a, Steven A. Titus a, Ruili Huang a, Douglas S. Auld a, James Inglese a, Christopher P. Austin a,Stephen W. Michnick b, Menghang Xia a, Craig J. Thomas a,*

a NIH Chemical Genomics Center, National Human Genome Research Institute, NIH 9800 Medical Center Drive, MSC 3370 Bethesda, MD 20892-3370, USAb Département de Biochimie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC, Canada H3C 3J7c Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH Bethesda, MD 20892-3370, USA

10a r t i c l e i n f o a b s t r a c t

Article history:Received 1 December 2008Revised 16 January 2009Accepted 20 January 2009Available online xxxx

Keywords:PDE4PDE4 inhibitorTriazolothiadiazinesTriazolopyridazinesAsthmaCOPD

0960-894X/$ - see front matter � 2009 Published bydoi:10.1016/j.bmcl.2009.01.057

* Corresponding author. Tel.: +1 301 217 4079; faxE-mail address: [email protected] (C.J. Thomas)

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An expansion of structure–activity studies on a series of substituted 7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadi-azine PDE4 inhibitors and the introduction of a related [1,2,4]triazolo[4,3-b]pyridazine based inhibitor ofPDE4 is presented. The development of SAR included strategic incorporation of known substituents onthe critical catachol diether moiety of the 6-phenyl appendage on each heterocyclic core. From thesestudies, (R)-3-(2,5-dimethoxyphenyl)-6-(4-methoxy-3-(tetrahydrofuran-3-yloxy)phenyl)-7H-[1,2,4]tria-zolo[3,4-b][1,3,4]thiadiazine (10) and (R)-3-(2,5-dimethoxyphenyl)-6-(4-methoxy-3-(tetrahydrofuran-3-yloxy)phenyl)-[1,2,4]triazolo[4,3-b]pyridazine (18) were identified as highly potent PDE4A inhibitors.Each of these analogues was submitted across a panel of 21 PDE family members and was shown to be highlyselective for PDE4 isoforms (PDE4A, PDE4B, PDE4C, PDE4D). Both 10 and 18 were then evaluated in diver-gent cell-based assays to assess their relevant use as probes of PDE4 activity. Finally, docking studies withselective ligands (including 10 and 18) were undertaken to better understand this chemotypes ability tobind and inhibit PDE4 selectively.

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RRThe second messenger cyclic 3,5-adenosine monophosphate

(cAMP) is a key regulator of numerous signaling cascades.1,2 Assuch, the production of cAMP by adenylate cyclase (AC) and thedegradation of cAMP by phosphodiesterases (PDEs) are highly reg-ulated. There are 11 primary families of PDEs (designated PDE1–PDE11) expressed throughout the human body, with several fami-lies having multiple isoforms. Much effort has been devoted tounderstanding the physiological role of each PDE isoform, andthe pharmacological inhibition of select PDE isoforms has proventherapeutically beneficial for several indications.2–4 The PDE4 fam-ily is comprised of 4 primary gene products (PDE4A, PDE4B, PDE4C,PDE4D) and is highly expressed in neutrophils and monocytes, CNStissue and smooth muscles of the lung.2–4 Not surprisingly, theclinical utility of PDE4 inhibitors has focused on asthma, chronicobstructive pulmonary disease (COPD), memory enhancementand as a general modulator of inflammation.4

At present, there are numerous chemotypes known to inhibitPDE4 and several of these inhibitors are currently being evaluatedin clinical settings.5 Reported molecules include rolipram (1), roflu-milast (2) and cilomilast (3). It is notable that each of these small

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bourdis, A. P. et al., Bioorg. M

molecules contains a common catechol diether motif. Crystallo-graphic studies of PDE4 bound to several of these derivatives haveshown the catechol diether forming a key hydrogen bond with aconserved glutamine residue (Gln443 in PDE4B and Gln369 inPDE4D).4,6,7 The choice of alkyl, cycloalkyl and heterocyclic ethermoieties is presumably driven by improvements to the potencyof each molecule and selected DMPK properties. It is noteworthythat methoxy, cyclopentyloxy, cyclopropylmethoxy and 2-difluo-romethoxy are repeatedly incorporated into known PDE4 inhibitorscaffolds. Another common ether substituent, an O-3-tetrahydrof-uranyl such as in 4, is found in numerous published and patentedchemotypes.8–10 The frequency with which these moieties areincorporated on small molecules intended to down-regulatePDE4 is compelling. However, there are few reports that directlycompare these substituents on the same core chemotype to gainan appreciation of each moieties potential and limitations.

Recently, we introduced a series of substituted 7H-[1,2,4]triaz-olo[3,4-b][1,3,4]thiadiazines as potent PDE4 inhibitors including5.11 Our initial 77 member matrix library revealed that the 3,4-catachol diether motif on the 6-phenyl appendage on the triazolo-thiadiazine ring system played a key role in defining thischemotype’s pharmacophore. Here, we systematically incorporatethe methoxy, cyclopentyloxy, cyclopropylmethoxy, 2-difluorome-

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S

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NN

OMeMeO

OMe

O

O

FF O

N N

Cl

ClO

MeONH

O O

MeO

CO2H

CN

1 (rolipram) 2 (roflumilast) 3 (cilomilast)

5

NN

MeO

O

O

OMe4

MeO

Figure 1. Structures of several known PDE4 inhibitors.

Q3

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thoxy and O-3-tetrahydrofuranyl moieties onto the 6-phenyl-triaz-olothiadiazine core in an attempt to improve potency, but alsobetter understand these frequently utilized groups. The DMPKprofiles of a representative triazolothiadiazine showed low micro-somal stability (rat) [high intrinsic clearance (Clint >300 lL/min/mg protein) and short half-life (t1/2 � 3 min)]. The removal/alter-ation of aromatic methoxy groups has been shown to positivelyaffect microsomal stability in several instances given the propen-sity for demethylation and glucuronidation of this functionalgroup. We were further interested in transposing the substitutionpatterns found on the triazolothiadiazine ring to the related triazo-lopyridazine ring system in order to remove the lone sulfur atomand eliminate S-oxidation as an additional mechanism of metabo-lism and clearance (Fig. 1).

The synthesis of substituted 7H-[1,2,4]triazolo[3,4-b][1,3,4]thi-adiazines was accomplished via previously reported methods (forfull synthetic details associated with the synthesis of substituted7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines see the Supportinginformation).11 The key condensation reaction between appropri-ately substituted 2-bromo-1-phenylethanone (ultimately the phe-nyl ring at the C6 position of the heterocycle) and appropriatelysubstituted 4-amino-3-phenyl-1H-1,2,4-triazole-5(4H)-thione(ultimately the phenyl ring at the C3 position of the heterocycle)was accomplished in ethanol at elevated temperatures. Utilizingthe known SAR from our previously reported matrix library, weopted to maintain the 2,5-dimethoxy substitution pattern on the3-phenyl appendage on the heterocyclic core. To incorporate thecyclopentyloxy, cyclopropylmethoxy, 2-difluoromethoxy andO-3-tetrahydrofuranyl moieties unto the 2-bromo-1-phenyletha-none precursor, we relied upon 1-(3-hydroxy-4-methoxy-

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Table 1PDE4A inhibition by compounds 5–10, 17 and 18

Analogue # R

S

NN

NN

R1

R2

OMe

5-10

MeO 5 (NCGC116) –6 –7 –8 –

NN

NN

R1

R2

OMe

MeO

17,18

9 –10 –17 –18 –

*Data represents the results from three separate experiments. Definitions: OCH3 = mdifluoromethoxy, O(3-THF) = O-3-tetrahydrofuranyl [racemic, RorS enantiomers].

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phenyl)ethanone as an orthogonally protected starting reagent.For the cyclopentyloxy and cyclopropylmethoxy substituents, weutilized a nucleophilic displacement of the corresponding alkylbromides to ultimately provide the substitution pattern found inderivatives 6 and 7 (Table 1). Reaction of 1-(3-(cyclopentyloxy)-4-methoxyphenyl)ethanone with dodecane-1-thiol in sodiummethoxide/DMF at 100 �C provided demethylation in a mildmanner.12 Treatment of the resulting 1-(3-(cyclopentyloxy)-4-hydroxyphenyl)ethanone with sodium 2-chloro-2,2-difluoroace-tate in DMF at 100 �C afforded the incorporation of the2-difluoromethoxy functionality on the C4 position of the catacholmoiety (found in derivative 8).13 Mitsonobu conditions wereutilized to condense tetrahydrofuran-3-ol (both racemic and R)with 1-(3-hydroxy-4-methoxyphenyl)ethanone to provide ana-logues 9 and 10.

The synthesis of [1,2,4]triazolo[4,3-b]pyridazines was basedupon the precedented works of Tišler and coworkers, Greenblattand coworkers and Street and coworkers.14–16 The general methodis outlined in Scheme 1 and begins with the coupling ofcommercially available 2,5-dimethoxybenzoic acid (11) and 3-chloro-6-hydrazinylpyridazine (12) to provide N0-(6-chloropyrida-zin-3-yl)-2,5-dimethoxybenzohydrazide (13) in good yields. Directtreatment of 13 with POCl3 at elevated temperature afforded thecyclization to core [1,2,4]triazolo[4,3-b]pyridazine ring system(analogue 14) and provided a common analogue for entry intothe convergent syntheses of multiple products via end-stage Suzu-ki–Miyaura couplings. Here, we were primarily interested in exam-ining the biochemical viability of this alternate heterocycle andlimited our explorations to the appropriately substituted O-3-tet-rahydrofuranyl derivatives. Boronic acids 15 and 16 were synthe-sized independently utilizing the aforementioned Mitsonobuprotocols and displacement of an aryl bromide with boronic acid.Following purification of 15 and 16, standard Suzuki–Miyaura con-ditions with microwave irradiation produced the appropriatelysubstituted [1,2,4]triazolo[4,3-b]pyridazines 17 and 18 in goodyields.

PDE4A inhibition profile. With several new analogues now inhand, we evaluated their inhibitory potency against PDE4A via apreviously reported, purified enzyme fluorescence polarizationassay (IMAP; Molecular Devices, CA).11 The results for the newlysynthesized 7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines 5–10 andthe novel [1,2,4]triazolo[4,3-b]pyridazines 17 and 18 are shownin Table 1. For 7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines 5–10,each substitution pattern yielded a molecule with potency in thelow nanomolar range. The enantiomerically pure O-(3-THF)[R]substitution of 10 showed the best potency with an IC50 value of

1 R2 PDE4A IC50 (nM)

OCH3 –OCH3 6.7 ± 0.4OCypent –OCH3 13 ± 0.8OCH2Cyprop –OCH3 6.1 ± 0.9OCH2Cyprop –OCHF2 11 ± 0.7

O(3-THF)[rac] –OCH3 3.4 ± 0.4O(3-THF)[R] –OCH3 3.0 ± 0.2O(3-THF)[S] –OCH3 7.3 ± 3.8O(3-THF)[R] –OCH3 1.5 ± 0.7

ethoxy, OCypent = cyclopentyloxy, OCH2Cyprop = cyclopropylmethoxy, OCHF2 = 2-

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F170

180

190

200

210

220

230

17

NNCl

NH

HN

O

MeO

OMe

OMe O

OH

OMe

NN

NN

MeO

OMeCl

NN

NN

OMe

MeO

MeO

OO

NN

NN

OMe

MeO

MeO

OO

MeO

B(OH)2

OO

MeO

B(OH)2

OO

iii

iii

N

N

NHH2N

Cl

i ii

11 12 13

14

15

16

18

Scheme 1. Reagents and conditions: (i) CDI, DMF, rt 2 h; (ii) POCl3, 105 oC, 2 h; (iii) 15 or 16, Pd(PPh3)4, 2 M aq. Na2CO3, DME, lwave, 90 �C 30 min.

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3.0 nM. As a result, the enantiomerically pure O-(3-THF)[R] and O-(3-THF)[S] substitutions were incorporated onto the [1,2,4]triazol-o[4,3-b]pyridazine core structure and the resulting constructswere found to have excellent potencies for PDE4A inhibition (IC50

value of 7.3 ± 3.8 nM for 17 and 1.5 ± 0.7 nM for 18). Several ana-logues were also explored with varying substitutions on the phenylring attached to the C3 position of the 1,2,4-triazole ring system.Substitutions included methoxy, fluoro, chloro and trifluoromethylgroups on the ortho, meta and para positions of the phenyl ring (forsynthetic details and characterization of these analogues see Sup-porting Information section). Analysis of these analogues con-firmed that various substitutions at one of the ortho positionswere necessary to maintain potent PDE4A inhibition (see Support-ing information). Additional substitutions have been shown to betolerated without effect on the inhibition of PDE4A.

Selectivity panel of PDE isoforms. Having arrived at several com-pounds with good potency profiles and divergent core heterocy-cles, it was of interest to confirm the selectivity of these agentsagainst a panel of PDE isoforms. BPS Bioscience (San Diego, CA)17

has available a panel of 21 PDE isoforms from all 11 primary PDEfamilies except PDE6 available for activity profiling. We submitted5, 10, 18 and 1 for analysis across this panel and the resulting IC50

determinations are shown in Table 2.It is apparent that both 10 and 18 represent novel inhibitors of

five isoforms of PDE4 with sub-nanomolar potencies found forPDE4A1A. The modest activities found for PDE3B and PDE10A1represent a sizeable enough difference in potency that cellularphenotypes will not be complicated by dual activities. Interest-ingly, PDE10 is considered to be an important modulator of cGMPregulation in the brain. Additionally, 10 and 18 may represent goodstarting points for optimizing compounds with potency for PDE10.This PDE family is considered to be an important regulator of cGMPin the brain, and to data few reports of potent and selective PDE10inhibitors have surfaced.

The identification of novel enzyme inhibitors via screening inpurified enzyme assays provides an important means for the iden-tification of small molecules with a known mechanism of action.However, not all active small molecules found in such screens find

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EDutility in cell-based assays for a myriad of reasons. To evaluate

whether the substantial in vitro inhibition of PDE4 by 10 and 18could be replicated in living cells we pursued two divergent, cell-based assays of PDE4 activity.

Cyclic-nucleotide gated ion channel cell-based assay. The first cell-based analysis of PDE4 activity utilized a recently reported assaybased on the coupling of a constitutively activated G-protein cou-pled receptor (GPCR) and cyclic-nucleotide gated (CNG) ion chan-nel coexpressed in HEK293 cells.18 The read-out for this assay isbased on measurement of membrane electrical potential by a po-tential-sensitive fluorophore (ACTOneTM dye kit). Inhibitors ofPDE4 will interfere with the native enzymatic conversion of cAMPto AMP resulting in increased intracellular levels of the cyclicnucleotide due to constitutive activity of the GPCR. In responseto increased amounts of cAMP, the CNG ion channel opens result-ing in membrane polarization. The dye reacts to this alteration inmembrane polarity with an increase in fluorescence detectableby fluorescence spectroscopy of whole cells read on a fluorescencemicrotitre plate reader.

Compounds 5, 10 and 18 were used in this analysis as alongwith the common PDE4 inhibitor 1, and the results are shown inFigure 2. In this assay, 1 was noted to produce an effective response(EC50) (registered as% activity) of 131.5 nM. In comparison, thetriazolothiadiazine based inhibitors were found to be more potentin this cell-based assay with 5 and 10 registering an EC50 value of18.7 and 2.3 nM, respectively. The EC50 of the lone triazolopyrid-azine 18 was 34.2 nM.

Protein-fragment complementation (PCA) cell-based assay. PCAstake advantage of the ability of well-engineered protein fragmentsto form a functional monomer with measurable enzymatic activitywhen brought into suitable proximity by interacting proteins towhich the fragments are fused.20,21 For our purposes, we utilizeda previously reported PCA based on reporter enzyme Renilla reni-formis luciferase (Rluc) N- and C-terminal fragments of Rluc fusedto the catalytic subunits (Cat) and inhibiting regulatory subunits(Reg) of protein kinase A (PKA).19 The signaling cascades initiatedby GPCR activation is mediated by cAMP production and activationof numerous protein kinases.1,20 Negative regulation of these

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Table 2PDE isoform selectivity data for 1, 5, 10 and 18

PDE isoform* 1 IC50/% inh. 5 IC50/%inh. 10 IC50/%inh. 18 IC50/%inh.

O

ONH

O

1

PDE1A Inactive Inactive 36% 32%PDE 1B Inactive Inactive 52% 56%PDE1C Inactive 26% 49% 74%

5S

NN

NN

OMeMeO

OMe

MeOPDE2A Inactive 41% 68% 54%PDE3A Inactive 1.7 lM 56% 54%PDE3B Inactive 720 nM 4.6 lM 2.3 lMPDE4A1A 102 nM 12.9 nM 0.26 nM 0.6 nMPDE4B1 901 nM 48.2 nM 2.3 nM 4.1 nM

S

NN

NN

OMeO

OMe

MeOO

10

PDE4B2 534 nM 37.2 nM 1.6 nM 2.9 nMPDE4C1 40% 452 nM 46 nM 106 nMPDE4D2 403 nM 49.2 nM 1.9 nM 2.1 nMPDE5A1 Inactive 60% 58% 51%PDE7A Inactive 73% 48% 59%

NN

NN

OMeO

OMe

MeOO

18

PDE7B Inactive 33% 43% 35%PDE8A1 Inactive 57% Inactive InactivePDE9A2 Inactive Inactive Inactive InactivePDE10A1 Inactive 823 nM 632 nM 388 nMPDE11A4 Inactive Inactive Inactive Inactive

* Data generated by BPS Biosciences (CA) [http://www.bpsbioscience.com] and represents either the IC50 value or the % inhibition at 10 lM of compound.

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events is the sole domain of the phosphodiesterase class of en-zymes.2 One ubiquitous pathway is activated when cAMP triggersthe disassociation of the PKA catalytic and regulatory subunits,which in turn, enables numerous signaling events. In the Rluc

UN

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RR

E

O

ONH

O

1

S

NN

NN

OMeO

OMe

MeOO

10

%A

ctiv

ity

Log [1], M

Log [10], M

%A

ctiv

ity

%A

ctiv

ity

%A

ctiv

ity

Figure 2. Cell-based analysis of novel PDE4 inhibitors. Effect of PDE4 inhibition by 1, 5Specific EC50 values are as follows: (A) EC50 for 1 = 131.5 nM. (B) EC50 for 5 = 18.7 nM. (C)four separate experiments.

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PCA PKA reporter, the regulatory subunit II beta cDNA is fusedthrough a sequence coding for a flexible polypeptide linker of 10amino acids (Gly.Gly.Gly.Gly.Ser)2 to the N-terminal fragment (RlucF[1])[amino acids 1–110 of Rluc] and the cDNA of the PKA catalytic

5

NN

NN

OMeO

OMe

MeOO

S

NN

NN

OMeMeO

OMe

MeO

18

Log [18], M

Log [5], M

, 10 and 18 in a cell-based cyclic nucleotide-gated cation channel biosensor assay.EC50 for 10 = 2.3 nM. (D) EC50 for 18 = 34.2 nM. The data represents the results from

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300

310

320

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subunit alpha is fused through the same flexible linker to the C-ter-minal fragment (Rluc F[2])[amino acids 111–311 of Rluc]. Theresulting constructs are designated Reg-F[1] and Cat-F[2] andreconstitute enzymatic activity of Rluc in the absence of cAMP Ithas been recently demonstrated that this assay could be used todetect the effects of PDE4 inhibition on PKA activation downstreamof basal b-2 adrenergic receptor (b2AR) activities.19

Here, we evaluated the effects of 1, 10 and 18 in HEK293 cellsstably expressing the b2AR and transiently transfected with therequired PKA-Rluc fragments [Reg-F[1] and CatF-[2]]. It was con-firmed that isoproterenol (19) activation of the b2AR is able toreduce luminescence (indicating dissociation of the Rluc biosensorcomplex and consequent activation of PKA catalytic activity)(Fig. 3A). Further, pretreatment with the selective b2AR inverseagonist IC118551 (20; decrease of basal b2AR activity) was capableof preventing the effects of 19 as was previously shown.19 Theseimportant controls confirm that alterations of the luminescencesignal are primarily mediated through the actions of the b2AR sig-naling to PKA. Further, the effect of 1 confirms the responsivenessof the assay to PDE4 inhibition. Treatment with 10 and 18 at100 lM and 10 lM concentrations displayed marked loss of lumi-nescence highly suggesting a b2AR mediated increase of cAMP dueto inhibition of PDE4 (Fig. 3B). Next, we examined the real-timekinetics of PKA subunit dissociation by administering 10 at a10 lM concentration. The shown real-time kinetics are normalizedon the control experiment of administering 10 following pretreat-ment with 1 lM of the inverse b2AR agonist 20. In four indepen-dent experiments, the presence of 10 reduced the luminescenceof the cell-based system by 25% to 50% within 2 min of administra-tion (Fig. 3C).

Docking of 10 at PDE4B. Given the potency, selectivity and cell-based PDE4 inhibition results for this chemotype, it was of interestto examine its binding modality to the PDE4 structure. The PDEclasses of enzymes are comprised of an N-terminal domain, acatalytic domain and a C-terminal domain. Crystallographic analy-ses of several PDE isozymes have aided researchers in understand-ing the divergent activities and pharmacology of this class ofproteins.6,23 Importantly, structures of PDE4 have been reportedin complex to AMP and several small molecule inhibitors.24–28

Through these efforts it was shown that the three domains ofPDE4 are coordinated through interactions with two metal cations(Zn2+ and Mg2+).7 The residues that coordinate these metals arehighly conserved across the PDE family. Both the Zn2+ and Mg2+

play important roles in the catalytic mechanism of cAMP hydroly-sis by coordinating the phosphate moiety. Other important insights

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RegF[1] + CatF[2] Reg

19

20

1

%of

PK

Aac

tiva

ti on

%of

PK

Aac

tiva

ti on

control

moc

k

Figure 3. Effect of PDE inhibition on b2AR regulated PKA activities in vivo. Bioluminescentransfected with the PKA reporter Reg-F[1]:Cat-F[2]2 (from three independent experimen(1 lM) and treatment with 1 (100 lM; 30 min) or 19 (1 lM, 30 min) on the on thConcentration dependent effect of 18, 10 and a related triazolothiadiazine that possessesindependent triplicates). Percentage of PKA activation is normalized based uponexperiment = pretreatment with 1 lM 20) of Reg-F[1]:Cat-F[2] dissociation in response

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include the recognition of a conserved glutamine residue (Q443 inPDE4B) that serves as an important binding residue for the purinemotif of cAMP and cGMP.

From the myriad of known PDE4 inhibitors, one common struc-tural motif that continues to be exploited are catachol diether-basedsmall molecules.4 Indeed, the structure of 1 formed the basis fornumerous catachol diether based inhibitors of PDE4 and the visual-ization of 1 bound to PDE4B provided the rational for its potent inhi-bition profile. From numerous crystallographic analyses andmodeling efforts it is clear that the catachol diether based inhibitorsbind to the catalytic domain of PDE4 through specific hydrogenbonds with the conserved glutamine residue. Our initial SAR explo-rations of triazolothiadiazine based PDE4 inhibitors confirmed that a3,4-dimethoxy phenyl moiety linked to the C6 position of the 3,6-dihydro-2H-1,3,4-thiadiazine ring was a crucial substitution patternfor potent PDE4 inhibition.11 Interestingly, the phenyl ring attachedto the C3 position of the 1,2,4-triazole ring system was found to bemore amendable to random substitutions without loss of function.This formed the basis for our supposition that these novel PDE4inhibitors were binding in a similar pose to that of 1.

To explore this hypothesis, we conducted docking simulationsusing the AutoDock software.29 We first retrieved the three-dimen-sional coordinates for PDE4B from the Protein Data Bank (PDB ID:1XMY). Protein and ligand structures were prepared in AutoDock29

and previously reported PDE4-inhibitor complexes were taken intoaccount when preparing the active site grid box. Flexibility wasgranted to the active site glutamine and the ligand(s). Followingmultiple docking simulations the most favorable binding conforma-tions were extracted based upon calculated binding constants(reported as Ki values and found to be in the low nanomolar rangefor favorable docking orientations). The primary docking modalityfor 10 is shown in Fig. 4. Importantly, this docking orientation is con-sistent with the idea that the catachol diether forms an integralhydrogen bond with Q443 (right panel) and the aromatic moiety isclearly positioned between the conserved isoleucine (I410) andphenylalanine (F446). The remainder of the molecule is shown toextend into the catalytic domain in close proximity to both theZn2+ and Mg2+ cations. Such an orientation would block theapproach of cAMP to the catalytic domain and forms the basis forinhibiting PDE4. This docking orientation is consistent with theknown SAR for this chemotype whereby the 3,4-dimethoxy phenylmoiety at the C6 position of the 3,6-dihydro-2H-1,3,4-thiadiazinering is crucial for maintaining inhibition in the low nanomolar rangewhereas the opposite phenyl ring is more amendable to changewithout significant loss of potency. It also demonstrates that altera-

100 µM10 µM

F[1] + CatF[2]

Time (sec)

%of

PK

Aac

tiva

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18 10

+ 10 µM 10

ce in A–C was detected from stable b2AR-HEK293 (see reference19) cells transientlyts). (A) Effect of combinations of pretreatment with the selective b2AR-antagonist 20e association of Reg-F[1]:Cat-F[2] (mean ± SD from independent triplicates). (B)no PDE4 inhibition on the association of Reg-F[1]:Cat-F[2] (30 min, mean ± SD from

20 (1 lM) pretreated cells. (C) Real-time kinetics (normalized on the controlto 10 treatment (10 lM, four independent samples).

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OO

F

340

350

360

370

380

390

Q1

400

410

Figure 4. Model of PDE4B and docking of 10. The left panel details the entire PDE4B structure (N-terminal domain, a catalytic domain and a C-terminal domain) bound to 10.The right panel shows the catalytic domain bound to 10 including interactions with conserved glutamine (Q443) isoleucine (I410) and phenylalanine (F446) and the Zn2+

(grey) and Mg2+ (green) cations. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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tions of the core heterocycle from the general triazolothiadiazinestructure to the triazolopyridazine structure will have limited affecton the inhibitory profile of these reagents. These docking poses mayallow future SAR explorations to focus on improved pharmacoki-netic and drug metabolism properties without compromisingpotency, efficacy and selectivity for this chemotype.

Numerous chemotypes exist for the potent, selective inhibitionof PDE4. Here, we expand upon our discovery of substituted 6-(3,4-dialkoxyphenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines andintroduce 6-(3,4-dialkoxyphenyl)-[1,2,4]triazolo[4,3-b]pyridazinesas novel inhibitors of PDE4. In this study, we examined the com-mon structural substitutions found on other catachol diether basedinhibitors of PDE4 and several of the resulting analogues areamong the most potent inhibitors of this important cellular target.These include methoxy, cyclopentyloxy, cyclopropylmethoxy, 2-difluoromethoxy and O-3-tetrahydrofuranyl moieties. It was foundthat the chirally pure R-O-3-tetrahydrofuranyl substitution main-tained the best potency in this study. Further, these reagents pos-sess impressive selectivity for PDE4 versus other PDE familymembers. However, these chemotypes, like others, do not possessubtype selectivity across PDE4 isozymes.4

Our initial analysis of the compounds and their activity wasbased upon an in vitro analysis using purified PDE4 protein. It iscritical to examine chemical probes discovered via purified-proteinassays within cell-based contexts to confirm activity and establishthat they are relevant for cell-based experimentation. Here, weexamine selected analogues (5, 10 and 18) in two different cell-based assays. One assay is based upon the ability of PDE4 to reducecAMP levels in a CNG cell line while the other utilizes a PCA repor-ter for PKA activity. Both analyses demonstrated the utility of thesenovel reagents as cell-based chemical probes of PDE4 activity.

Finally, with the myriad of structural data surrounding PDE4 andPDE4 complexes with selected inhibitors, it was important toexplore the binding modality of these compounds. Docking studiesdemonstrated that these agents utilize the conserved binding modewhereby the catachol diether functionality forms a strong interac-tion with a conserved glutamine residue. This docking orientationfurther provides a roadmap for additional SAR around the seeminglymodifiable phenyl ring attached to the 1,2,4-triazole moiety of thecore heterocycle. This key aspect of these reagents may be of impor-tance during attempts to modify ADME properties of these com-pounds without altering the affinity or selectivity for PDE4.

PDE4 inhibitors are highly sought after as probes of selected cellsignalling pathways and as potential therapeutics in diverse areasincluding memory enhancement and COPD. Here, we expand on

Please cite this article in press as: Skoumbourdis, A. P. et al., Bioorg. M

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PRthe potential of substituted triazolothiadiazines and introduce tria-

zolopyridazines as potent and selective inhibitors of this importantcellular target. Not only are selected analogues of this novel chem-otype capable of down-regulating purified isozymes of PDE4, butthey maintain excellent cell-based activity as well. Their bindingmodality is predicted to mimic known catachol diether basedinhibitors of PDE4. Importantly, both computational and structureactivity studies suggest that the phenyl ring at the C3 position ofthe 1,2,4-triazole ring system could be modified providing a mech-anism for advanced SAR considerations.

Uncited reference

Ref. [22].

Acknowledgments

We thank Ms. Allison Peck for critical reading of this manu-script. This research was supported by the Molecular LibrariesInitiative of the National Institutes of Health Roadmap for MedicalResearch, the Intramural Research Program of the National HumanGenome Research Institute and the National Institute of Dental andCraniofacial Research.

Supplementary data

Experimental methods for the cyclic nucleotide-gated cationchannel assay, protein-fragmentation complementation assaysand molecular docking and the full synthetic procedures and char-acterization of reported compounds are detailed. This material isavailable free of charge via the Internet. Supplementary data asso-ciated with this article can be found, in the online version, atdoi:10.1016/j.bmcl.2009.01.057.

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